High temperature sterilization. Sterilization of laboratory glassware Chemical sterilization method

Sterilization is represented by physical, chemical, mechanical and biological methods and various methods. The feasibility of using a particular sterilization method and its methods depends on the characteristics of the material to be sterilized, its physical and chemical properties. The duration of sterilization depends on the object being sterilized, the sterilizing agent and its dose, the temperature and humidity of the environment.

Physical sterilization method Methods of physical sterilization include drying, burning and calcination, boiling, pasteurization and tindization, hot air (dry heat), ultrasound, ultraviolet and radioactive radiation, high frequency current, sunlight. The most common method of sterilizing items that can be exposed to high temperatures is sterilization with fire, hot air and saturated steam under pressure. Fire is used to burn infected objects that do not represent any value (unnecessary papers, old wallpaper, rags, garbage), to disinfect the sputum of tuberculosis patients, the corpses of people and animals who died from particularly dangerous infections, as well as to burn and calcinate various objects . Burning and calcination are widely used in microbiological practice for the disinfection of instruments, laboratory and pharmaceutical glassware. Calcination in a burner flame or flambéing is a method of sterilization in which the object is completely sterilized, as vegetative cells, cysts and spores of microorganisms die. Typically, loops, spatulas, pipettes, slides and coverslips, small instruments and other contaminated items are sterilized by calcination if they cannot be boiled. It is not recommended to sterilize scissors and scalpels by heating, as the cutting surface becomes dull when exposed to fire. One of the simplest and most common methods of physical sterilization used in medical practice is hot air sterilization (dry heat). Dry heat sterilization is carried out in drying ovens (Pasteur ovens). Dry hot air has a bactericidal, virusicidal, sporicidal effect and is used mainly for the sterilization of glass products (laboratory glassware - Petri dishes, flasks, pipettes, test tubes, etc.), as well as metal products that can be sterilized with steam under pressure. In addition, dry heat is used to sterilize items made of porcelain and heat-resistant substances (talc, white clay), as well as mineral and vegetable oils, fats, petroleum jelly, lanolin, and wax. The most effective mode for this sterilization method, which ensures the death of vegetative forms and spores, is a temperature of 160 - 180 degrees for 15 minutes. You cannot sterilize food items, isotonic solution, or items made of rubber and synthetic materials with dry heat, as liquids boil and pour out, and rubber and synthetic materials melt. Sterilization with saturated steam under pressure is the most reliable and most often used method for sterilizing dressings, water, some medicines, culture media, soft equipment, instruments, as well as for disinfecting waste contaminated material. In surgical practice, dressings, surgeons' gowns, and underwear for the operated patient are disinfected using steam in autoclaves. Steam sterilization under pressure is carried out in special devices - autoclaves. Autoclaving completely destroys all microorganisms and spores. The steam pressure sterilization method is based on heating the material with saturated water vapor under pressure above atmospheric pressure. The combined action of high temperature and steam makes this method particularly effective. In this case, both vegetative cells and microbial spores die. Microbial spores die within 10 minutes under the influence of saturated water vapor, and vegetative forms die within 1 to 4 minutes. The high bactericidal power of saturated steam is due to the fact that, under the influence of water vapor under pressure, the proteins of the microbial cell swell and coagulate, as a result of which the microbial cells die. The bactericidal effect of saturated water vapor is enhanced by excess pressure. Sterilization in an autoclave is carried out under different modes. Thus, simple nutrient media (meat - peptone agar and meat - peptone broth) are sterilized for 20 minutes at 120 degrees (1 atm.). But with this mode it is impossible to sterilize media containing proteins, carbohydrates and other substances that are easily changed by heating. Media with carbohydrates are sterilized in an autoclave at 0.5 atm. 10 – 15 minutes or fractionally flowing steam. Using high temperature, you can destroy the most persistent forms of pathogenic microorganisms (including spore-forming ones) not only on the surface of the objects being disinfected, but also in their depth. This is the great advantage of high temperature as a reliable means of sterilization. However, some items deteriorate under the influence of high temperatures and in these cases it is necessary to resort to other methods and means of disinfection. Complete sterilization of materials and objects that do not allow the use of high temperature sterilization is achieved by repeated sterilization with water vapor in a Koch apparatus at a temperature not exceeding 100 degrees. This method is called fractional sterilization. It boils down to the fact that the remaining unkilled spore forms of microbes, after a day in a thermostat at 37 degrees, germinate into vegetative cells, the death of which occurs during the subsequent sterilization of this object with flowing steam. Treatment with fluid steam is carried out three times for 30–40 minutes. Heating the material once at a temperature below 100 degrees is known as pasteurization. Pasteurization was proposed by Pasteur and is intended mainly to destroy mostly non-spore microorganisms. Pasteurization is carried out at 60 - 70 degrees for 15 to 30 minutes, at 80 degrees for 10 to 15 minutes. In microbiological practice, pasteurization seed material often used to isolate pure cultures of spore-forming microorganisms and to determine the ability of microorganisms to form spores. For liquids that lose taste and other valuable qualities when exposed to high temperatures (milk, berry and fruit juices, beer, nutrient media containing carbohydrates or urea, etc.), sterilization with flowing steam is carried out at 50 - 60 degrees for 15 - 33333330 minutes or at 70 - 80 degrees for 5 - 10 minutes. In this case, microbes of average resistance die, while more resistant microbes and spores are preserved. Fractional 5-6-fold sterilization at 60 degrees for 1 hour is called tyndalization. Many medical products made from polymer materials, do not withstand sterilization steam method according to generally accepted modes. For many products, due to the characteristics of the liquids they contain (preservatives, medicines and other products), it is impossible to sterilize using generally accepted methods and methods. For such products, individual sterilization regimes are developed to ensure reliable sterilization of objects. Thus, sterilization of the rotor to separate blood into fractions is carried out with water vapor at a temperature of 120 degrees for 45 minutes. Sterility of preservative containers is achieved at 110 degrees for 60 minutes. Boiling is a sterilization method used to desterilize reusable syringes, surgical instruments, rubber tubes, glass and metal utensils. Sterilization by boiling is carried out in sterilizers. Spore forms in boiling water die after 20 - 30 minutes. Boiling for 45 minutes is widely used to disinfect secretions and other infectious materials, linen, dishes, toys, and patient care items. Hot water (60 - 100 degrees) with detergents is used when washing and cleaning to mechanically remove dirt and microorganisms. Most vegetative cells die at 70 degrees after 30 minutes. Filtration sterilization is used in cases where substrates cannot withstand heating, in particular for media containing proteins, serums, some antibiotics, vitamins, and volatile substances. This technique is quite widely used for sterilizing a culture liquid, when it is necessary to free it from microbial cells, but to preserve all the metabolic products it contains unchanged. The method involves filtering liquids through special filters that have finely porous partitions and therefore retain microbial cells. The two most widely used types of filters are membrane filters and Seitz filters. Membrane filters are prepared from collodion, acetate, cellulose and other materials. Seitz filters are made from a mixture of asbestos and cellulose. In addition, filters made of kaolin with an admixture of quartz sand, infusor earth and other materials (“candles” by Chamberlan, Berkfeld) are used for sterilization. Membrane and asbestos filters are designed for one-time use. With ultraviolet irradiation, the bactericidal effect is provided by rays with a length of 200 - 450 nm, the source of which is bactericidal lamps. With the help of bactericidal lamps, air is sterilized with ultraviolet rays in medical and preventive institutions, microbiological laboratory boxes, and enterprises Food Industry, in boxes for the production of vaccines and serums, in operating rooms, manipulation rooms, children's institutions, etc. Ultraviolet rays have high antimicrobial activity and can cause the death of not only vegetative cells, but also their spores. Sunlight causes the death of microorganisms as a result of the action ultraviolet irradiation and drying. Drying with sunlight has a detrimental effect on many types of microorganisms, but its effect is superficial and therefore sunlight plays a supporting role in sterilization practice. Recently, in the treatment of wounds and burns, coatings made of synthetic and natural polymers have been used in the form of gels. Polymer antiseptic films are widely used for local treatment of wounds and burns. They contain such broad-spectrum antimicrobial agents as catapol, dioxidine, blue iodine, as well as sorbitol containing glutaraldehyde. To sterilize these films, ionizing radiation is used at a dose of 20.0 kGy. During the industrial production of polymer antiseptic films and sorbents, their sterility under this sterilization regime is fully ensured. Radioactive radiation kills all types of microorganisms, both in vegetative and spore forms. It is widely used for sterilization in enterprises producing sterile products and sterile disposable medical devices, for the disinfection of wastewater and raw materials of animal origin.

Mechanical sterilization method Mechanical sterilization methods remove germs from the surface of objects. These include washing, shaking out, sweeping, wet wiping, airing, ventilation, vacuuming, washing.

Chemical method sterilization Plastics are now increasingly used in medical practice. They are used in dentistry, maxillofacial surgery, traumatology, orthopedics, and surgery. Most plastics cannot withstand the heat sterilization methods of steam under pressure and dry heat (dry heated air). The solutions of alcohol, diocide, and ternary solution used to sterilize such objects do not ensure the sterility of the products being processed. Therefore, gas and radiation methods, as well as solutions of chemicals, are used to sterilize plastic products. The introduction into the practice of medical institutions of a large number of products made from thermolabile materials contributes to the introduction of radiation, gas methods disinfection and sterilization with disinfectant solutions. During chemical sterilization, gases and agents from various chemical groups are used (peroxide, phenolic, halogen-containing, aldehydes, alkalis and acids, surfactants, etc.). For everyday use, detergents, cleaning, bleaching and other preparations are produced that have an antimicrobial effect due to the introduction of various chemicals into their composition. These preparations are used for cleaning and disinfecting sanitary equipment, dishes, linen, etc. Formaldehyde steam (steamform) can be used in medical institutions for sterilization metal products medical purposes (scalpels, needles, tweezers, probes, clamps, hooks, wire cutters, etc.). Before sterilization with formaldehyde vapor, products must be subjected to pre-sterilization cleaning and thoroughly dried. When sterilized in any way chemically the regulations for processing a particular object depend on the characteristics of the object being disinfected, the resistance of microbes, and the characteristics of the properties chemical preparation, ambient temperature, humidity and other factors. Thus, the sterility of metal instruments is achieved after five hours of storage in a sealed chamber with steam at a temperature of at least 20 degrees and a relative humidity of 95 - 98%; at a temperature of 15 degrees, complete sterility of these objects is achieved only after 16 hours. The sporicidal activity of glutaraldehyde depends on temperature. Its optimum action occurs at a temperature of 15 – 25 degrees. As the temperature rises, the sporicidal activity of this drug decreases. Chemical sterilization is used to a somewhat limited extent. Most often, this method is used to prevent bacterial contamination of culture media and immunobiological preparations (vaccines and serums). Substances such as chloroform, toluene, and ether are most often added to nutrient media. If it is necessary to free the medium from these preservatives, it is heated in a water bath at 56 degrees and the preservatives evaporate. To preserve vaccines or serums, merthiolate, boric acid, and formalin are used.

Biological sterilization method Biological sterilization is based on the use of antibiotics. This method is widely used in the cultivation of viruses.

Sterilization

Sterilization is sterilization, i.e. complete liberation of environmental objects from microorganisms and their spores.

Sterilization is carried out in various ways:

1) physical (exposure to high temperature, UV rays, use bacterial filters);

2) chemical (use of various disinfectants, antiseptics);

3) biological (use of antibiotics).

In laboratory practice, physical methods of sterilization are usually used.

The possibility and feasibility of using one or another sterilization method is determined by the characteristics of the material to be sterilized, its physical and chemical properties.

Physical methods

Calcination in a burner flame or flambéing is a method of sterilization in which the object is completely sterilized, since both vegetative cells and microbial spores die. Typically, bacteriological loops, spatulas, pipettes, slides and cover glasses, and small instruments are calcined. Scissors and scalpels should not be sterilized by heating, since under the influence of fire the cutting surface becomes dull.

Dry heat sterilization

Sterilization with dry heat or hot air is carried out in Pasteur ovens (drying dry-heat ovens). The Pasteur oven is a double-walled cabinet made of heat-resistant materials - metal and asbestos. Heat the cabinet with gas burners or electric heating devices. Electrically heated cabinets are equipped with regulators that ensure required temperature. To control the temperature, there is a thermometer inserted into the hole in the top wall of the cabinet.

Liquids (nutrient media, isotonic sodium chloride solution, etc.), items made of rubber and synthetic materials cannot be sterilized with dry heat, since liquids boil and pour out, and rubber and synthetic materials melt.

Sterilization by boiling

Boiling is a sterilization method that guarantees sterility provided there are no spores in the sterilized material. Used for processing syringes, instruments, glass and metal utensils, rubber tubes, etc. Steam sterilization under pressure is carried out in an autoclave. This sterilization method is based on exposing the materials being sterilized to saturated water vapor at a pressure above atmospheric. As a result of such sterilization, both vegetative and spore forms of microorganisms die with a single treatment. An autoclave (Fig. 12) is a massive boiler, covered on the outside with a metal casing, hermetically sealed with a lid, which is tightly screwed to the boiler with hinged bolts.

The temperature and duration of autoclaving of nutrient media is determined by their composition specified in the recipe for preparing the nutrient medium. For example, simple media (meat-peptone agar, meat-peptone broth) are sterilized for 20 minutes at 120 ° C (1 atm). However, at this temperature it is impossible to sterilize media containing native proteins, carbohydrates and other substances that are easily changed by heating. Media with carbohydrates are sterilized fractionally at 100°C or in an autoclave at 112°C (0.5 atm) for 10-15 minutes. Various liquids, devices with rubber hoses, plugs, bacterial candles and filters are sterilized for 20 minutes at 120 ° C (1 atm).

Sterilization with flowing steam is carried out in a Koch apparatus. This method is used in cases where the object being sterilized changes at a temperature above 100° C. Nutrient media containing urea, carbohydrates, milk, potatoes, gelatin, etc. are sterilized with flowing steam.

The Koch apparatus (boiler) is a metal cylinder lined on the outside (to reduce heat transfer) with felt or asbestos. The cylinder is closed with a conical lid with a hole for steam to escape. Inside the cylinder there is a stand, to the level of which water is poured. A bucket with a hole is placed on the stand into which the material to be sterilized is placed. The Koch apparatus is heated using gas or electricity. The sterilization time is counted from the moment of vigorous steam release at the edges of the lid and from the steam outlet. Sterilize for 30-60 minutes. At the end of sterilization, heating is stopped. Remove the bucket of material from the apparatus and leave it at room temperature until the next day. Warming is carried out for 3 days in a row at a temperature of 100° C for 30-60 minutes. This method is called fractional sterilization. During the first heating, vegetative forms of microbes die, while spore forms are preserved. Within a day, the spores manage to germinate and turn into vegetative forms, which die on the second day of sterilization. Since it is possible that some of the spores did not have time to germinate, the material is kept for another 24 hours, and then a third sterilization is carried out. Sterilization with flowing steam in a Koch apparatus does not require special control, since the sterility of the prepared nutrient media is an indicator of the correct operation of the device. You can also sterilize with flowing steam in an autoclave with the lid unscrewed and the outlet valve open.

Sterilization by ultraviolet irradiation

Sterilization with UV rays is carried out using special installations - bactericidal lamps. UV rays have high antimicrobial activity and can cause the death of not only vegetative cells, but also spores. UV irradiation is used to sterilize air in hospitals, operating rooms, children's institutions, etc. In a microbiological laboratory, a box is treated with UV rays before work.

Chemical methods

This type of sterilization is used to a limited extent, and it serves mainly to prevent bacterial contamination of culture media and immunobiological preparations (vaccines and serums).

Substances such as chloroform, toluene, and ether are most often added to nutrient media. If it is necessary to free the medium from these preservatives, it is heated in a water bath at 56 ° C (the preservatives evaporate).

To preserve vaccines and serums, use merthiolate. boric acid, formalin, etc.

Biological sterilization

Biological sterilization is based on the use of antibiotics. This method is used for cultivating viruses.

B. Detailed technology for the production of cattle whey in the slaughterhouses of Lyon

Blood has already been drawn from 1,000 animals, the serum has been bottled and distributed free of charge to almost 20,000 children.

Thus it is shown that industrial production whey at the slaughterhouse in compliance with aseptic rules and sanitary regulations is possible.

The serotherapy enterprise does not provide additional guarantees - it does not have the right to perform an autopsy on the donor animal.

The technology used in our production may seem less strict from an aseptic point of view than the classical method. But it has a big advantage in speed, since the serum is completely produced on the day of blood collection.

If current circumstances have prompted us to produce the serum in a slaughterhouse, it is clear that this is a temporary measure, since hematogen and medicinal serums can only be produced in a specialized institute.

Animal selection. In Lyon, Dr. Guier, the chief veterinarian of the slaughterhouse, and Dr. Fontenay, the veterinary inspector, themselves select donors from among the livestock intended to provide our city with meat. The selected animal is branded on the right shoulder to facilitate further control.

After slaughter, the animals' organs are carefully checked. It is known that autopsy is the most reliable method of detecting tuberculosis.

Subsequent operations will show that the serum of the diseased animal was never consumed.

Taking blood from animals. In the blood collection room, the donor bull is securely held by an automatic yoke.

The veterinarian disinfects the skin with iodine at the level of the animal's neck and makes an incision in the area of the jugular vein with a scalpel. Venous puncture is performed with a trocar sterilized by prolonged boiling. Once blood appears, an autoclaved rubber hose is attached to the trocar to connect it directly and aseptically to the defibrinator.

The defibrinating technology and method for sterilizing the defibrinator are described below.

From each animal, 8-10 liters of blood are obtained, which is weighed on scales located under the apparatus.

Sterilization methods

To facilitate the sanitary control operation, each defibrinator has a label with the data of the donor animal.

It should be noted that blood sampling is carried out aseptically thanks to a closed circuit of all components: trocar, rubber hose and defibrinator, which are pre-sterilized.

A label with the date of blood collection accompanies the collected blood from the moment of venous puncture until it is converted into serum and stored in refrigerators.

Blood defibration. In most serotherapy establishments, blood poured into glass vessels separates the serum under the pressure of the load. Under these conditions, blood first obtained from cattle contains little serum (approximately 10%).

Therefore, the serotherapy center in Lyon uses special technology, giving 50% of the whey, and also in a shorter time.

Dr. Merrier was able to develop this method partly after the findings he made at the Royal Institute in Rotterdam and at the Serotherapeutic Institute in Milan.

Once blood is obtained from these institutes, it is defibrinated in sterile machines that resemble churns.

Within 5 minutes, the blood is collected in a vessel protected from air. It is necessary to strictly observe the defibration time: if it is insufficient, coagulation can occur, and if it is too much, hemolysis can occur (due to the rupture of red blood cells). It is necessary to use a laboratory clock that allows you to mark exactly 15 minutes of defibrination.

At the top of Fig. 4 shows that the number of blood draws has reached 1000, which is recorded on the Center’s emblem.

Centrifugation.

Immediately after defibration, the devices are transferred to the laboratory, located a few meters from the blood collection room. The contents of each defibrinator are processed separately so that the serum of an animal that turns out to be sick can be removed.

Blood that does not clot after defibrination is passed through an Alfa Laval separator (a milk separator that we have adapted for the production of whey).

Under the influence of centrifugal force, the blood is divided into equal parts: the red part contains red balls, and the transparent part makes up the serum (fibrin remains on the defibrinator blades).

From one animal, 8-10 liters of blood or approximately 4-5 liters of serum are obtained, which is poured into a Pyrex bottle, sterilized at 180°C.

A defibrinator label is attached to the bottle, and a card is filled out under the same number to ensure sanitary control.

A special antiseptic is added to the serum, designed so that it is sufficiently active without disturbing the transparency and taste of the serum. For 1 liter of serum, also add 100 ml of a solution containing 1:1000 formaldehyde and 1:5000 syunuxol.

(Between preparations, the centrifuge is thoroughly disinfected with an antiseptic.)

In principle, the red fraction of the blood should be returned to the butchers to make blood sausage, but most often it remains unused, so syrup can be prepared from it using the technology described below.

Special cases of preparing blood syrup. The syrup has two advantages: it makes it possible to use the red part of the blood remaining after receiving the serum, and it has a pleasant taste that children like.

Due to the lack of glycerin, it is difficult to prepare syrup for long-term storage, but you can make a very active product in water and give it to children 2-3 tablespoons a day. Add 20% to the red part of the blood drinking water and store the syrup in glaciers while sanitary control is carried out.

Then add an equal part 100% sugar syrup (sugar can be obtained from the cards of the groups to whom the syrup is intended).

Lemon or orange extract is added to the syrup to neutralize the taste of blood and poured into 250 ml bottles.

Tyndallization of whey.

Immediately after centrifugation, that is, less than an hour after blood collection, the serum is lysed at 56°C for an hour.

To do this, it is lowered into a water bath with an automatically maintained temperature. Tyndallization at this temperature (at a higher temperature the whey coagulates) is necessary for partial sterilization of the whey, although the speed of its preparation in itself is a guarantee of asepsis.

It should be noted that each five-liter bottle is provided with the original defibrinator label, thus the numbering of the serum corresponds to the numbering of the donor animals.

Control card file. The card allows you to check at any time the origin of the donor animal, the stages of whey preparation, the bottling date, as well as the distribution of the whey.

Sanitary control. While the vessels are stored in the refrigeration chamber, veterinarian inspectors are engaged in sanitary control of donor animals. After their slaughter, a thorough autopsy is carried out to reveal the slightest symptoms of tuberculosis.

If a disease is detected, the corresponding serum can be easily withdrawn. It is known that the blood of each animal is processed separately and the serum is contained in separate, numbered bottles.

Serotherapy center Public Committee childhood hygiene at Lyon slaughterhouses

Control number …

Bovine whey in liters

Card and label

The above card and label prevents any confusion.

In Lyon, sanitary measures are especially strict, as the same veterinarians select the animals, draw blood and check the meat.

Sterilization of equipment. Animal serum is an excellent breeding ground for microbes and can only be partially sterilized. At temperatures above 56° they coagulate and become cloudy if a strong antiseptic is added. Therefore, during all serum production operations, maximum sterility is required; equipment must be decontaminated before use.

Individual defibrinators are sterilized in the following way: the night before blood collection, they are filled with an antiseptic solution, and a few hours before blood collection, they are emptied of antiseptic using a tap located in the lower part of the device. Centrifuges used to produce serum are also treated with an antiseptic, including between processing the contents of each defibrinator.

All glassware, including five-liter vessels for storing whey, is sterilized in an electric oven at a temperature of 180°C.

250 ml bottles for serum are also sterilized at 180°C. To simplify the operation, the dishes are located in boxes, which are used during bottling and distribution to the public.

flower delivery to Voskresensk

Sterilization is represented by physical, chemical, mechanical and biological methods and various methods.

The feasibility of using a particular sterilization method and its methods depends on the characteristics of the material to be sterilized, its physical and chemical properties.

The duration of sterilization depends on the object being sterilized, the sterilizing agent and its dose, the temperature and humidity of the environment.

Physical sterilization method

Methods of the physical method of sterilization include drying, burning and calcination, boiling, pasteurization and tindalization, hot air (dry heat), ultrasound, ultraviolet and radioactive radiation, high frequency current, sunlight.

The most common method of sterilizing items that can be exposed to high temperatures is sterilization with fire, hot air and saturated steam under pressure.

Fire is used to burn infected objects that do not represent any value (unnecessary papers, old wallpaper, rags, garbage), to disinfect the sputum of tuberculosis patients, the corpses of people and animals who died from particularly dangerous infections, as well as to burn and calcinate various objects .

Burning and calcination are widely used in microbiological practice for the disinfection of instruments, laboratory and pharmaceutical glassware.

Calcination in a burner flame or flambéing is a method of sterilization in which the object is completely sterilized, as vegetative cells, cysts and spores of microorganisms die.

Typically, loops, spatulas, pipettes, slides and coverslips, small instruments and other contaminated items are sterilized by calcination if they cannot be boiled. It is not recommended to sterilize scissors and scalpels by heating, as the cutting surface becomes dull when exposed to fire.

One of the simplest and most common methods of physical sterilization used in medical practice is hot air sterilization (dry heat). Dry heat sterilization is carried out in drying ovens (Pasteur ovens). Dry hot air has a bactericidal, virusicidal, sporicidal effect and is used mainly for the sterilization of glass products (laboratory glassware - Petri dishes, flasks, pipettes, test tubes, etc.), as well as metal products that can be sterilized with steam under pressure.

In addition, dry heat is used to sterilize items made of porcelain and heat-resistant substances (talc, white clay), as well as mineral and vegetable oils, fats, petroleum jelly, lanolin, and wax. The most effective mode for this sterilization method, which ensures the death of vegetative forms and spores, is a temperature of 160 - 180 degrees for 15 minutes.

You cannot sterilize food items, isotonic solution, or items made of rubber and synthetic materials with dry heat, as liquids boil and pour out, and rubber and synthetic materials melt.

Sterilization with saturated steam under pressure is the most reliable and most often used method for sterilizing dressings, water, some medicines, culture media, soft equipment, instruments, as well as for disinfecting waste contaminated material.

In surgical practice, dressings, surgeons' gowns, and underwear for the operated patient are disinfected using steam in autoclaves. Steam sterilization under pressure is carried out in special devices - autoclaves.

Autoclaving completely destroys all microorganisms and spores. The steam pressure sterilization method is based on heating the material with saturated water vapor under pressure above atmospheric pressure. The combined action of high temperature and steam makes this method particularly effective. In this case, both vegetative cells and microbial spores die.

Microbial spores die within 10 minutes under the influence of saturated water vapor, and vegetative forms die within 1 to 4 minutes.

The high bactericidal power of saturated steam is due to the fact that, under the influence of water vapor under pressure, the proteins of the microbial cell swell and coagulate, as a result of which the microbial cells die.

The bactericidal effect of saturated water vapor is enhanced by excess pressure.

Sterilization in an autoclave is carried out under different modes.

Thus, simple nutrient media (meat - peptone agar and meat - peptone broth) are sterilized for 20 minutes at 120 degrees (1 atm.). But with this mode it is impossible to sterilize media containing proteins, carbohydrates and other substances that are easily changed by heating.

Media with carbohydrates are sterilized in an autoclave at 0.5 atm. 10 – 15 minutes or fractionally flowing steam.

Using high temperature, you can destroy the most persistent forms of pathogenic microorganisms (including spore-forming ones) not only on the surface of the objects being disinfected, but also in their depth.

This is the great advantage of high temperature as a reliable means of sterilization. However, some items deteriorate under the influence of high temperatures and in these cases it is necessary to resort to other methods and means of disinfection.

Complete sterilization of materials and objects that do not allow the use of high temperature sterilization is achieved by repeated sterilization with water vapor in a Koch apparatus at a temperature not exceeding 100 degrees. This method is called fractional sterilization. It boils down to the fact that the remaining unkilled spore forms of microbes, after a day in a thermostat at 37 degrees, germinate into vegetative cells, the death of which occurs during the subsequent sterilization of this object with flowing steam.

Treatment with fluid steam is carried out three times for 30–40 minutes. Heating the material once at a temperature below 100 degrees is known as pasteurization. Pasteurization was proposed by Pasteur and is intended mainly to destroy mostly non-spore microorganisms. Pasteurization is carried out at 60 - 70 degrees for 15 to 30 minutes, at 80 degrees for 10 to 15 minutes.

In microbiological practice, pasteurization of seed material is often used to isolate pure cultures of spore-forming microorganisms and to identify the ability of microorganisms to form spores.

For liquids that lose taste and other valuable qualities when exposed to high temperatures (milk, berry and fruit juices, beer, nutrient media containing carbohydrates or urea, etc.), sterilization with flowing steam is carried out at 50 - 60 degrees for 15 - 33333330 minutes or at 70 - 80 degrees for 5 - 10 minutes. In this case, microbes of average resistance die, while more resistant microbes and spores are preserved.

Fractional 5-6-fold sterilization at 60 degrees for 1 hour is called tyndalization.

Many medical products made from polymer materials cannot withstand steam sterilization according to generally accepted regimes. For many products, due to the characteristics of the liquids they contain (preservatives, medicines and other products), it is impossible to sterilize using generally accepted methods and methods. For such products, individual sterilization regimes are developed to ensure reliable sterilization of objects.

Thus, sterilization of the rotor to separate blood into fractions is carried out with water vapor at a temperature of 120 degrees for 45 minutes.

Sterility of preservative containers is achieved at 110 degrees for 60 minutes.

Boiling is a sterilization method used to desterilize reusable syringes, surgical instruments, rubber tubes, glass and metal utensils.

Sterilization by boiling is carried out in sterilizers. Spore forms in boiling water die after 20 - 30 minutes. Boiling for 45 minutes is widely used to disinfect secretions and other infectious materials, linen, dishes, toys, and patient care items.

Hot water (60 - 100 degrees) with detergents is used when washing and cleaning to mechanically remove dirt and microorganisms.

Most vegetative cells die at 70 degrees after 30 minutes.

Filtration sterilization is used in cases where substrates cannot withstand heating, in particular for media containing proteins, serums, some antibiotics, vitamins, and volatile substances. This technique is quite widely used for sterilizing a culture liquid, when it is necessary to free it from microbial cells, but to preserve all the metabolic products it contains unchanged.

The method involves filtering liquids through special filters that have finely porous partitions and therefore retain microbial cells.

The two most widely used types of filters are membrane filters and Seitz filters.

Membrane filters are prepared from collodion, acetate, cellulose and other materials.

Seitz filters are made from a mixture of asbestos and cellulose.

In addition, filters made of kaolin with an admixture of quartz sand, infusor earth and other materials (“candles” by Chamberlan, Berkfeld) are used for sterilization.

Membrane and asbestos filters are designed for one-time use.

With ultraviolet irradiation, the bactericidal effect is provided by rays with a length of 200 - 450 nm, the source of which is bactericidal lamps.

With the help of bactericidal lamps, air is sterilized with ultraviolet rays in medical and preventive institutions, boxes in microbiological laboratories, in food industry enterprises, in boxes for the production of vaccines and serums, in operating rooms, manipulation rooms, children's institutions, etc.

Ultraviolet rays have high antimicrobial activity and can cause the death of not only vegetative cells, but also their spores.

Sunlight causes the death of microorganisms as a result of ultraviolet irradiation and drying.

Drying with sunlight has a detrimental effect on many types of microorganisms, but its effect is superficial and therefore sunlight plays a supporting role in sterilization practice.

Recently, in the treatment of wounds and burns, coatings made of synthetic and natural polymers have been used in the form of gels.

Polymer antiseptic films are widely used for local treatment of wounds and burns. They contain such broad-spectrum antimicrobial agents as catapol, dioxidine, blue iodine, as well as sorbitol containing glutaraldehyde. To sterilize these films, ionizing radiation is used at a dose of 20.0 kGy. During the industrial production of polymer antiseptic films and sorbents, their sterility under this sterilization regime is fully ensured.

Radioactive radiation kills all types of microorganisms, both in vegetative and spore forms. It is widely used for sterilization in enterprises producing sterile products and sterile disposable medical devices, for the disinfection of wastewater and raw materials of animal origin.

Mechanical sterilization method

Mechanical sterilization methods remove germs from the surface of objects. These include washing, shaking out, sweeping, wet wiping, airing, ventilation, vacuuming, washing.

Chemical sterilization method

Plastics are now increasingly used in medical practice.

They are used in dentistry, maxillofacial surgery, traumatology, orthopedics, and surgery. Most plastics cannot withstand the heat sterilization methods of steam under pressure and dry heat (dry heated air). The solutions of alcohol, diocide, and ternary solution used to sterilize such objects do not ensure the sterility of the products being processed.

Therefore, gas and radiation methods, as well as solutions of chemicals, are used to sterilize plastic products.

The introduction of a large number of products made of thermolabile materials into the practice of medical institutions contributes to the introduction of radiation and gas methods of disinfection and sterilization with disinfectant solutions.

During chemical sterilization, gases and agents from various chemical groups are used (peroxide, phenolic, halogen-containing, aldehydes, alkalis and acids, surfactants, etc.). For everyday use, detergents, cleaning, bleaching and other preparations are produced that have an antimicrobial effect due to the introduction of various chemicals into their composition.

These preparations are used for cleaning and disinfecting sanitary equipment, dishes, linen, etc.

Formaldehyde steam (steamform) can be used in medical institutions to sterilize metal medical products (scalpels, needles, tweezers, probes, clamps, hooks, wire cutters, etc.).

Before sterilization with formaldehyde vapor, products must be subjected to pre-sterilization cleaning and thoroughly dried.

When sterilizing by any chemical method, the procedure for processing a particular object depends on the characteristics of the object being disinfected, the resistance of microbes, the characteristics of the properties of the chemical, ambient temperature, humidity and other factors.

Thus, the sterility of metal instruments is achieved after five hours of storage in a sealed chamber with steam at a temperature of at least 20 degrees and a relative humidity of 95 - 98%; at a temperature of 15 degrees, complete sterility of these objects is achieved only after 16 hours.

The sporicidal activity of glutaraldehyde depends on temperature. Its optimum action occurs at a temperature of 15 – 25 degrees. As the temperature rises, the sporicidal activity of this drug decreases.

Chemical sterilization is used to a somewhat limited extent. Most often, this method is used to prevent bacterial contamination of culture media and immunobiological preparations (vaccines and serums). Substances such as chloroform, toluene, and ether are most often added to nutrient media. If it is necessary to free the medium from these preservatives, it is heated in a water bath at 56 degrees and the preservatives evaporate.

To preserve vaccines or serums, merthiolate, boric acid, and formalin are used.

Biological sterilization method

Biological sterilization is based on the use of antibiotics.

This method is widely used in the cultivation of viruses.

Sterilization (from Latin sterilis - sterile) involves the complete inactivation of microbes on objects being processed.

Pasteur oven - dry heat sterilization.

There are three main methods of sterilization: heat, radiation, chemical.

Iodine.

Heat sterilization is based on the sensitivity of microbes to high temperature.

At 60 °C and the presence of water, denaturation of proteins occurs, including enzymes, as a result of which the vegetative forms of microbes die. Spores containing a very small amount of bound water and having dense shells are inactivated at 160-170 °C. For heat sterilization, dry heat and steam under pressure are mainly used.
Dry heat sterilization is carried out in dry heat ovens, or Pasteur ovens. The Pasteur oven is a tightly closed metal cabinet, heated by electricity and equipped with a thermometer.

Disinfection of the material in it occurs at 160-170 °C for 60-120 minutes. The disadvantage of this method is that only some sterilizable objects, such as laboratory glass, can withstand such high temperatures.
The most universal method of sterilization is steam treatment under pressure in autoclaves, in which dressings, linen, many instruments, culture media, solutions, infectious material, etc. are sterilized.

An autoclave is a metal cylinder with strong walls, hermetically sealed, consisting of a water-steam and sterilizing chamber. The device is equipped with a pressure gauge, thermometer and other monitoring devices. Increased pressure is created in the autoclave, which leads to an increase in the boiling point of water. So, at 0.5 atm the boiling point is 80 °C, at 1 atm - 100 °C, at 2 atm - 121 °C and at 3 atm - 136 °C.

Due to the fact that, in addition to high temperature, steam acts on microorganisms, spores die already at 120 ° C. The most common autoclave operating mode is 2 atm, 121 °C, 15-20 minutes. Sterilization time decreases with increasing atmospheric pressure, and therefore the boiling point. Microorganisms die in a few seconds, but the material is processed for a longer time, since, firstly, the temperature must be high inside the material being sterilized, and, secondly, there is a so-called safety field, designed for possible deviation from the specified parameters when autoclave operation.

Tags: body, growth, sterilization, enzyme

The life of microorganisms is closely dependent on environmental conditions. All environmental factors that influence microorganisms can be divided into three groups: physical, chemical and biological, the beneficial or detrimental effect of which depends both on the nature of the factor itself and on the properties of the microorganism.

Physical factors

Of the physical factors, temperature, drying, radiant energy, and ultrasound have the greatest influence on the development of microorganisms.

Temperature. The life activity of each microorganism is limited by certain temperature limits. This temperature dependence is usually expressed by three main points: minimum - the temperature below which the reproduction of microbial cells stops; optimum - best temperature for the growth and development of microorganisms; maximum - the temperature above which the vital activity of cells weakens or stops. The optimal temperature usually corresponds to the temperature conditions of the natural habitat.

All microorganisms in relation to temperature are divided into psychrophiles, mesophiles and thermophiles.

Psychrophiles (from the Greek psychros - cold, phileo - love), or cold-loving microorganisms, grow at relatively low temperatures: minimum temperature - 0° C, optimal - 10-20° C, maximum - 30° C. This group includes microorganisms living in northern seas and oceans, soil, wastewater. This also includes luminous and iron bacteria, as well as microbes that cause food spoilage in the cold (below 0° C).

Mesophiles (from the Greek mesos - middle) are the most extensive group, including most saprophytes and all pathogenic microorganisms. The optimal temperature for them is 28-37° C, the minimum is 10° C, the maximum is 45° C.

Thermophiles (from the Greek termos - heat, heat), or heat-loving microorganisms, develop at temperatures above 55 ° C, the temperature minimum for them is 30 ° C, the optimum is 50-60 ° C, and the maximum is 70-75 ° C. They found in hot mineral springs, the surface layer of soil, self-heating substrates (manure, hay, grain), and the intestines of humans and animals. Among thermophiles there are many spore forms.

High and low temperatures have different effects on microorganisms. Some are more sensitive to high temperatures. Moreover, the higher the temperature beyond the maximum, the faster the death of microbial cells occurs, which is due to the denaturation (coagulation) of cell proteins.

Vegetative forms of mesophilic bacteria die at a temperature of 60° C for 30-60 minutes, and at 80-100° C - after 1-2 minutes. Bacterial spores are much more resistant to high temperatures. For example, spores of anthrax bacilli can withstand boiling for 10-20 minutes, and spores of clostridium botulism - 6 hours. All microorganisms, including spores, die at a temperature of 165-170 ° C for an hour (in a dry-heat oven) or when exposed to steam under pressure 1 atm (in an autoclave) for 30 minutes.

The effect of high temperatures on microorganisms is the basis of sterilization - the complete release of various objects from microorganisms and their spores (see below).

Many microorganisms are extremely resistant to low temperatures. Salmonella typhus and Vibrio cholerae survive for a long time in ice. Some microorganisms remain viable at liquid air temperatures (-190°C), and bacterial spores can withstand temperatures down to -250°C.

Only individual species pathogenic bacteria are sensitive to low temperatures (for example, Bordetella pertussis and parapertussis, Neisseria meningococcus, etc.). These properties of microorganisms are taken into account in laboratory diagnostics and when transporting the test material - it is delivered to the laboratory protected from cooling.

The action of low temperatures stops putrefactive and fermentation processes, which is widely used to preserve food in refrigeration units, cellars, and glaciers. At temperatures below 0° C, microbes fall into a state of suspended animation - metabolic processes slow down and reproduction stops. However, if there are appropriate temperature conditions and nutrient medium, the vital functions of microbial cells are restored. This property of microorganisms is used in laboratory practice to preserve microbial cultures at low temperatures. Rapid changes in high and low temperatures (freezing and thawing) also have a detrimental effect on microorganisms - this leads to rupture of cell membranes.

Drying. Water is necessary for the normal functioning of microorganisms. Drying leads to dehydration of the cytoplasm, disruption of the integrity of the cytoplasmic membrane, as a result of which the nutrition of microbial cells is disrupted and their death occurs.

The timing of the death of different types of microorganisms under the influence of drying differs significantly. For example, pathogenic Neisseria (meningococci, gonococci), Leptospira, Treponema pallidum and others die when dried after a few minutes. Vibrio cholerae can withstand drying for 2 days, Salmonella typhoid - 70 days, and Mycobacterium tuberculosis - 90 days. But the dried sputum of tuberculosis patients, in which the pathogens are protected by a dry protein cover, remains infectious for 10 months.

Spores are particularly resistant to drying, as well as to other environmental influences. Spores of anthrax bacilli retain the ability to germinate for 10 years, and spores of mold fungi for up to 20 years.

The unfavorable effect of drying on microorganisms has long been used for preserving vegetables, fruits, meat, fish and medicinal herbs. At the same time, when exposed to high humidity conditions, such products quickly deteriorate due to the restoration of microbial activity.

The freeze-drying method is widely used for storing cultures of microorganisms, vaccines and other biological preparations. The essence of the method is that microorganisms or preparations are first frozen and then dried under vacuum conditions. In this case, microbial cells enter a state of suspended animation and retain their biological properties for several months or years.

Radiant Energy. In nature, microorganisms are constantly exposed to solar radiation. Direct sunlight causes the death of many microorganisms within a few hours, with the exception of photosynthetic bacteria (green and purple sulfur bacteria). The harmful effects of sunlight are due to the activity ultraviolet rays(UV rays). They inactivate cell enzymes and damage DNA. Pathogenic bacteria are more sensitive to the action of UV rays than saprophytes. Therefore, it is better to store microbial cultures in the laboratory in the dark. In this regard, Buchner's experience is demonstrative.

In a Petri dish with thin layer agar is produced by abundant inoculation of any bacterial culture. Letters cut out of black paper are glued onto the outer surface of the seeded cup, forming, for example, the word “typhus”. The cup, with its bottom facing up, is irradiated by direct sunlight for 1 hour. Then the papers are removed, and the cup is placed in a thermostat at 37° C for a day. Bacterial growth is observed only in those places of the agar that were protected from UV rays by stickers letters. The rest of the agar remains transparent, i.e. there is no growth of microorganisms (Fig. 11).

The importance of sunlight as a natural factor in improving the health of the external environment is great. It frees the air, water of natural reservoirs, and upper layers of soil from pathogenic bacteria.

The bactericidal (bacteria-destroying) effect of UV rays is used to sterilize the air in closed spaces (operating rooms, dressing rooms, boxes, etc.), as well as water and milk. The source of these rays are ultraviolet radiation lamps and bactericidal lamps.

Other types of radiant energy - X-rays, α-, β-, γ-rays have a detrimental effect on microorganisms only in large doses, on the order of 440-280 J/kg. The death of microbes is caused by the destruction of nuclear structures and cellular DNA. Low doses of radiation stimulate the growth of microbial cells. Microorganisms are much more resistant to radioactive radiation than higher organisms. Thionic bacteria are known to live in deposits uranium ores. Bacteria were found in water from nuclear reactors at a concentration of ionizing radiation of 20-30 kJ/kg.

The bactericidal effect of ionizing radiation is used for the preservation of certain food products, sterilization of biological preparations (sera, vaccines, etc.), while the properties of the sterilized material do not change.

In recent years, disposable products such as polystyrene pipettes, Petri dishes, wells for serological reactions, syringes, as well as suture material - catgut, etc. have been sterilized using the radiation method.

Ultrasound causes significant damage to microbial cells. Under the influence of ultrasound, gases located in the liquid environment of the cytoplasm are activated, and high pressure arises inside the cell (up to 10,000 atm). This leads to rupture of the cell membrane and cell death. Ultrasound is used to sterilize food products (milk, fruit juices) and drinking water.

High pressure. Bacteria and especially their spores are resistant to mechanical pressure. In nature, bacteria are found that live in seas and oceans at a depth of 1000-10000 m under pressure from 100 to 900 atm. Some types of bacteria can withstand pressures of up to 3000-5000 atm, and bacterial spores - even 20,000 atm.

Chemical factors

The effect of chemicals on microorganisms varies depending on the nature of the chemical compound, its concentration, and the duration of exposure to microbial cells. Depending on the concentration, a chemical substance can be a source of nutrition or have an inhibitory effect on the vital activity of microorganisms. For example, a 0.5-2% glucose solution stimulates the growth of microbes, and 20-40% glucose solutions inhibit the proliferation of microbial cells.

Many chemical compounds that have a detrimental effect on microorganisms are used in medical practice as disinfectants and antiseptics.

Chemicals used for disinfection are called disinfectants. Disinfection refers to measures aimed at destroying pathogenic microorganisms in various environmental objects. Disinfectants include halide compounds, phenols and their derivatives, salts of heavy metals, some acids, alkalis, alcohols, etc. They cause the death of microbial cells, acting in optimal concentrations for a certain time. Many disinfectants have a harmful effect on the tissues of the macroorganism.

They are called antiseptics chemical substances, which can cause the death of microorganisms or inhibit their growth and reproduction. They are used for therapeutic purposes (chemotherapy), as well as for the disinfection of wounds, skin, and human mucous membranes. Hydrogen peroxide, alcohol solutions of iodine, brilliant green, solutions of potassium permanganate, etc. have antiseptic properties. Some antiseptic substances (acetic, sulfurous, benzoic acid, etc.) in doses that are harmless to humans are used for food preservation.

According to the mechanism of action, chemical substances with antimicrobial activity can be divided into several groups.

1. Surfactants (fatty acids, soaps and other detergents) cause a decrease in surface tension, which leads to disruption of the functioning of the cell wall and cytoplasmic membrane of microorganisms.

2. Phenol, cresol and their derivatives cause coagulation of microbial proteins. They are used to disinfect infectious material in microbiological practice and infectious diseases hospitals.

3. Oxidizing agents, interacting with microbial proteins, disrupt the activity of enzymes and cause protein denaturation. Active oxidizing agents are chlorine and ozone, which are used to disinfect drinking water. Chlorine derivatives (bleach, chloramine) are widely used for disinfection purposes. Hydrogen peroxide, potassium permanganate, iodine, etc. have oxidizing properties.

4. Formaldehyde is used in the form of a 40% solution (formalin) for disinfection. It kills vegetative and spore forms of microorganisms. Formalin blocks the amino groups of microbial cell proteins and causes their denaturation.

5. Salts of heavy metals (mercury, lead, zinc, gold, etc.) coagulate the proteins of the microbial cell, thereby causing their death. A number of metals (silver, gold, mercury, etc.) have a bactericidal effect on microorganisms in negligible concentrations. This property is called oligodynamic action (from the Latin oligos - small, dinamys - strength). It has been proven that water in silver vessels does not rot due to the bactericidal effect of silver ions. For the prevention of blenorrhea * newborns for a long time a 1% solution of silver nitrate was used. Colloidal solutions organic compounds silver (protargol, collargol) is also used in the form of local antiseptics.

* (Blennorea is an inflammation of the conjunctiva of the eye caused by gonococci.)

Mercury preparations have a strong antimicrobial effect. Since ancient times, mercury bichloride, or mercuric chloride (at a dilution of 1:1000), has been used for disinfection. However, she has toxic effect on the tissue of the macroorganism and its use is limited.

6. Dyes (diamond green, rivanol, etc.) have the property of inhibiting the growth of bacteria. Solutions of a number of dyes are used as antiseptics, and are also added to some nutrient media to inhibit the growth of accompanying microflora.

The destructive effect of a number of physical and chemical factors on microorganisms forms the basis of aseptic and antiseptic methods, widely used in medical and sanitary practice.

Asepsis is a system of preventive measures that prevent microbial contamination of an object (wounds, surgical field, cultures of microorganisms, etc.) based on physical methods.

Antiseptics is a set of measures aimed at destroying microorganisms in a wound, the whole body or on environmental objects, using various disinfecting chemicals.

Biological factors

In natural habitats, microorganisms do not exist in isolation, but are in complex relationships, which come down mainly to symbiosis, metabiosis and antagonism.

Symbiosis is the cohabitation of organisms of different species, bringing them mutual benefit. At the same time, together they develop better than each of them separately.

Symbiotic relationships exist between nodule bacteria and leguminous plants, between filamentous fungi and blue-green algae (lichens): The symbiosis of lactic acid bacteria and alcoholic yeast is used to prepare some lactic acid products (kefir, koumiss).

Metabiosis is a type of relationship in which the metabolic products of one type of microorganism create the necessary conditions for the development of others. For example, putrefactive microorganisms that break down protein substances contribute to the accumulation of ammonium compounds in the environment and create favorable conditions for the growth and development of nitrifying bacteria. And the development of anaerobes in well-aerated soil would be impossible without aerobes that absorb free oxygen.

Metabiotic relationships are widespread among soil microorganisms and underlie the cycle of substances in nature.

Antagonism is a form of relationship in which one microorganism inhibits the development of another or can cause its complete death. Antagonistic relationships have developed among microorganisms in the struggle for existence. Everywhere they live, there is a constant struggle between them for food sources, air oxygen, and habitat. Thus, most pathogenic bacteria, having entered the external environment (soil, water) with the secretions of patients, cannot withstand long-term competition with numerous saprophytes and die relatively quickly.

Antagonism can be caused by the direct influence of microorganisms on each other or by the action of their metabolic products. For example, protozoa devour bacteria, and phages lyse them. The intestines of newborns are colonized by lactic acid bacteria Bifidobacterium bifidum. By releasing lactic acid, they suppress the growth of putrefactive bacteria and thereby protect the still fragile organism of infants from intestinal disorders. Some microorganisms in the process of life produce various substances that have a detrimental effect on bacteria and other microbes. These substances include antibiotics (see "Antibiotics").

Control questions

1. What physical factors influence the life activity of microorganisms?

2. What substances are classified as disinfectants and how do they differ in their mechanism of action on microorganisms?

3. List what relationships exist between microorganisms?

Sterilization

Sterilization is sterilization, i.e. complete liberation of environmental objects from microorganisms and their spores.

Sterilization is carried out in various ways:

1) physical (exposure to high temperature, UV rays, use of bacterial filters);

2) chemical (use of various disinfectants, antiseptics);

3) biological (use of antibiotics).

In laboratory practice, physical methods of sterilization are usually used.

The possibility and feasibility of using one or another sterilization method is determined by the characteristics of the material to be sterilized, its physical and chemical properties.

Physical methods

Dry heat sterilization

Sterilization with dry heat or hot air is carried out in Pasteur ovens (drying ovens). The Pasteur oven is a double-walled cabinet made of heat-resistant materials - metal and asbestos. Heat the cabinet using gas burners or electric heating devices. Electrically heated cabinets are equipped with regulators to ensure the required temperature. To control the temperature, there is a thermometer inserted into the hole in the top wall of the cabinet.

Dry heat is used to sterilize laboratory glassware. The dishes prepared for sterilization are loosely loaded into the oven to ensure uniform and reliable heating of the material being sterilized. Close the cabinet door tightly, turn on the heating device, bring the temperature to 160-165 ° C and sterilize at this temperature for 1 hour. At the end of sterilization, turn off the heating, but do not open the cabinet door until the oven has cooled down; otherwise cold air, entering the cabinet can cause cracks in hot cookware.

Sterilization in a Pasteur oven can be carried out at different temperature conditions and exposure (sterilization time) (Table 1).

To control sterilization in a Pasteur oven, silk threads are moistened in a culture of spore-forming bacteria, dried, placed in a sterile Petri dish and placed in a Pasteur oven. Sterilization is carried out at a temperature of 165° C for 1 hour (for control, some of the threads are left at room temperature). Then the sterilized and control threads are placed on the surface of the agar in a Petri dish or placed in test tubes with broth and incubated in a thermostat at 37° C for 2 days. With proper operation of the Pasteur oven, there will be no growth in test tubes or dishes with nutrient media in which sterilized threads were placed, since bacterial spores will die, while bacterial spores on threads that were not sterilized (control) will germinate on nutrient media growth will be noted.

To determine the temperature inside the Pasteur oven, you can use sucrose or granulated sugar, which caramelizes at a temperature of 165-170 ° C.

Preparing laboratory glassware for sterilization in a Pasteur oven. Before sterilization, laboratory glassware (Petri dishes, graduated and Pasteur pipettes, vials, flasks, test tubes) must be thoroughly washed, dried and wrapped in paper, otherwise after sterilization they may again become contaminated with air bacteria.

Petri dishes are wrapped in paper one or more pieces at a time or placed in special metal cases.

Cotton swabs are inserted into the upper ends of the pipettes to prevent the test material from entering the mouth. Graduated pipettes are wrapped in long strips of paper 4-5 cm wide. The volume of the wrapped pipette is marked on the paper. In pencil cases, graduated pipettes are sterilized without additional wrapping in paper.

Note. If the graduation on the pipettes is poorly visible, it is restored before sterilization. Oil paint is applied to the pipette and, without allowing the paint to dry, barium sulfate powder is rubbed into it using a cloth. After this, remove excess paint with a rag, which remains only in the graduation notches. Pipettes treated in this way should be rinsed.

The sharp ends of Pasteur pipettes are sealed in a burner flame and wrapped in paper, 3-5 pieces at a time. Pasteur pipettes must be wrapped carefully so as not to break off the sealed ends of the capillaries.

Vials, flasks, test tubes are closed with cotton-gauze stoppers. The cork should fit into the neck of the vessel 2/3 of its length, not too tight, but not loose either. A paper cap is placed over the stoppers on each vessel (except test tubes). Test tubes are tied together in groups of 5-50 and wrapped with paper.

Note. At high temperatures, the paper in which cups and pipettes are wrapped, and cotton wool turn yellow and may even become charred, so every new variety paper received by the laboratory should be tested at the accepted temperature conditions.

Control questions

1. What is meant by the term sterilization?

2. How is sterilization carried out?

3. What is sterilized by calcination over fire?

4. Describe the structure and operating mode of the Pasteur oven.

5. What is sterilized in a Pasteur oven?

6. How are glassware prepared for sterilization?

7. Why can’t nutrient media and rubber objects be sterilized in a Pasteur oven?

Exercise

Prepare Petri dishes, graduated pipettes, Pasteur pipettes, test tubes, flasks and vials for sterilization.

Sterilization by boiling

Sterilization by boiling is usually carried out in a sterilizer - a metal box rectangular shape with a tight-fitting lid. The material to be sterilized is placed on the mesh available in the sterilizer and filled with water. To increase the boiling point and eliminate water hardness, add 1-2% sodium bicarbonate (it is better to use distilled water). The sterilizer is closed with a lid and heated. The beginning of sterilization is considered to be the moment of boiling of water, the boiling time is 15-30 minutes. At the end of sterilization, the mesh with instruments is removed by the side handles with special hooks, and the instruments in it are taken with sterile tweezers or forceps, which are boiled along with the rest of the instruments.

Steam sterilization is carried out in two ways: 1) steam under pressure; 2) flowing steam.

Pressure steam sterilization produced in an autoclave. This sterilization method is based on exposing the materials being sterilized to saturated water vapor at a pressure above atmospheric. As a result of such sterilization, both vegetative and spore forms of microorganisms die with a single treatment.

An autoclave (Fig. 12) is a massive boiler, covered on the outside with a metal casing, hermetically sealed with a lid, which is tightly screwed to the boiler with hinged bolts. Another, smaller diameter, which is called a sterilization chamber, is inserted into the outer boiler. Objects to be sterilized are placed in this chamber. Between both boilers there is free space, called a water vapor chamber. Water is poured into this chamber through a funnel fixed on the outside to a certain level marked on a special water-measuring tube. When water is boiled in a water-steam chamber, steam is produced. The sterilization chamber is equipped with an outlet cock with a safety valve to allow steam to escape when the pressure increases above the required level. A pressure gauge is used to determine the pressure created in the sterilization chamber.

Rice. 12. Autoclave diagram. M - pressure gauge; PC - safety valve; B - funnel for water; K 2 - tap for water release; K 3 - valve for steam release

Normal atmospheric pressure (760 mm Hg) is taken as zero. There is a certain relationship between the pressure gauge readings and temperature (Table 2).

Currently, there are autoclaves with automatic control of the operating mode. In addition to the usual pressure gauge, they are equipped electric contact pressure gauge, which prevents pressure from increasing above a given value and thereby ensures constant desired temperature in an autoclave.

Steam under pressure sterilizes various nutrient media (except those containing native proteins), liquids (isotonic sodium chloride solution, water, etc.); devices, especially those with rubber parts.

Attention! Infected material is also neutralized in autoclaves. Cups and test tubes containing cultures of microorganisms are placed in special metal buckets or tanks with holes in the lid for steam penetration and sterilized in an autoclave at 126 ° C (1.5 atm) for 1 hour. Instruments are sterilized in the same way after working with bacteria , forming disputes.

Only specially trained persons are allowed to work with the autoclave, who must strictly and accurately follow the rules specified in the instructions supplied with the device.

Autoclaving technique. 1. Before work, check the serviceability of all parts and the grinding of the taps.

2. Water (distilled or boiled to prevent scale formation) is poured through a funnel mounted outside the boiler to the top mark of the water meter glass. The tap under the funnel is closed.

3. The material to be sterilized is placed in the sterilization chamber on a special mesh. Items should not be loaded too tightly, as steam must pass freely between them, otherwise they will not heat up to the required temperature and may remain unsterile.

4. The rubber gasket on the lid is rubbed with chalk for better sealing.

5. The lid is closed and bolted to the autoclave body, and the bolts are screwed in pairs crosswise.

6. Open the outlet valve connecting the sterilization chamber with the outside air all the way, and begin to heat the autoclave. The autoclave is usually heated using gas or electricity.

When the autoclave is heated, the water boils, the resulting steam rises between the walls of the boilers and through special holes in the wall of the internal boiler (see Fig. 12), enters the sterilization chamber and exits through the open outlet valve. First, the steam escapes along with the air in the autoclave. It is necessary that all air is forced out of the autoclave, otherwise the pressure gauge reading will not correspond to the temperature in the autoclave.

The appearance of a continuous strong stream of steam indicates complete removal of air from the autoclave; After this, the outlet valve is closed and the pressure inside the autoclave begins to gradually increase.

7. The beginning of sterilization is considered the moment when the pressure gauge readings reach the specified value. Heating is adjusted so that the pressure in the autoclave does not change over a certain period of time.

8. After the sterilization time has expired, the heating of the autoclave is stopped, and the steam is released through the outlet valve. When the pressure gauge needle drops to zero, open the lid. To avoid burns from steam remaining in the autoclave, the lid should be opened towards you.

The temperature level in the autoclave, i.e. the correctness of the pressure gauge readings, can be checked. To do this, use various substances that have a certain melting point: antipyrine (113° C), resorcinol and sulfur (119° C), benzoic acid (120° C). One of these substances is mixed with a negligible amount of dye (muchsin or methylene blue) and poured into a glass tube, which is sealed and placed in a vertical position between the material to be sterilized. If the temperature is sufficient, the substance will melt and turn the color of the corresponding dye.

To check the effectiveness of sterilization, a test tube with a known spore culture is placed in the autoclave. After autoclaving, the tube is transferred to a thermostat for 24-48 hours, the absence or presence of growth is noted. Lack of growth indicates proper operation of the device.

Sterilization with flowing steam produced in the Koch apparatus. This method is used in cases where the object being sterilized changes at a temperature above 100° C. Nutrient media containing urea, carbohydrates, milk, potatoes, gelatin, etc. are sterilized with flowing steam.

The Koch apparatus (boiler) is a metal cylinder lined on the outside (to reduce heat transfer) with felt or asbestos. The cylinder is closed with a conical lid with a hole for steam to escape. Inside the cylinder there is a stand, to the level of which water is poured. A bucket with a hole is placed on the stand into which the material to be sterilized is placed. The Koch apparatus is heated using gas or electricity. The sterilization time is counted from the moment of vigorous steam release at the edges of the lid and from the steam outlet. Sterilize for 30-60 minutes. At the end of sterilization, heating is stopped. Remove the bucket of material from the apparatus and leave it at room temperature until the next day. Warming is carried out for 3 days in a row at a temperature of 100° C for 30-60 minutes. This method is called fractional sterilization. During the first heating, vegetative forms of microbes die, while spore forms are preserved. Within a day, the spores manage to germinate and turn into vegetative forms, which die on the second day of sterilization. Since it is possible that some of the spores did not have time to germinate, the material is kept for another 24 hours, and then a third sterilization is carried out. Sterilization with flowing steam in a Koch apparatus does not require special control, since the sterility of the prepared culture media is an indicator of the correct operation of the device. You can also sterilize with flowing steam in an autoclave with the lid unscrewed and the outlet valve open.

Control questions

1. What nutrient media are steam sterilized?

2. What is a sterilizer and how does it work?

3. Why should distilled water be used when sterilizing by boiling?

4. Describe the structure and operating mode of the autoclave.

5. What is sterilized in an autoclave?

6. What serves as a control for proper sterilization during autoclaving?

7. What is flowing steam sterilization?

8. Describe the structure of the Koch apparatus.

9. What is the purpose of fractional sterilization?

Exercise

Fill the form.

Fractional sterilization can also be carried out in a Koch coagulant.

Koch's coagulant is used to coagulate whey and egg culture media, and simultaneously with the compaction of the medium, it is sterilized.

Koch's coagulant is a flat metal box with double walls, coated on the outside with heat-insulating material. Water is poured into the space between the walls through a special hole located in the upper part of the outer wall. The hole is closed with a stopper into which a thermometer is inserted. The device is closed with two lids: glass and metal. Through the glass lid you can observe the coagulation process. Test tubes with media are placed on the bottom of the coagulator in an inclined position.

The coagulator is heated using gas or electricity. The media are sterilized once at a temperature of 90°C for 1 hour or fractionally - 3 days in a row at 80°C for 1 hour.

Tyndallization* - fractional sterilization at low temperatures - used for substances that are easily destroyed and denatured at a temperature of 60 ° C (for example, protein liquids). The material to be sterilized is heated in a water bath or in special devices with thermostats at a temperature of 56-58° C for an hour for 5 days in a row.

* (The sterilization method is named after Tyndall, who proposed it.)

Pasteurization- sterilization at 65-70 ° C for 1 hour, proposed by Pasteur to destroy non-spore forms of microbes. Milk, wine, beer, fruit juices and other products are pasteurized. Milk is pasteurized to remove lactic acid and pathogenic bacteria (brucella, mycobacterium tuberculosis, shigella, salmonella, staphylococcus, etc.). When pasteurizing beer, fruit juices, and wine, microorganisms that cause different kinds fermentation. Pasteurized foods are best kept refrigerated.

Control questions

1. What is the purpose and structure of the Koch coagulator?

2. What are the methods of sterilization in a clotting machine?

3. What is tyndalization?

4. What is pasteurization?

Sterilization by ultraviolet irradiation

Control questions

1. What properties do ultraviolet rays have?

2. In what cases is sterilization using ultraviolet radiation used?

Mechanical sterilization using bacterial filters

Filtration sterilization is used in cases where the objects being sterilized change when heated. Filtration is carried out using bacterial filters made from various fine-porous materials. The pores of filters must be small enough (up to 1 micron) to ensure mechanical retention of bacteria, therefore some authors refer to filtration as mechanical methods sterilization.

The filtration method is used to sterilize nutrient media containing protein, serum, and some antibiotics, and also to separate bacteria from viruses, phages and exotoxins.

In microbiological practice, Seitz asbestos filters, membrane filters and Chamberlant and Berkefeld filters (candles) are used.

Seitz filters are discs made from a mixture of asbestos and cellulose. Their thickness is 3-5 mm, diameter 35-140 mm. The domestic industry produces filters of two brands: “F” (filtering) - retaining suspended particles but allowing bacteria to pass through; "SF" (sterilizing) - with smaller pores, retaining bacteria, but allowing viruses through. Crumpled asbestos plates, as well as plates with breaks and cracks, are unsuitable for work.

Membrane filters are made from nitrocellulose. They are disks white 0.1 mm thick and 35 mm in diameter. Depending on the pore size, they are designated No. 1, 2, 3, 4 and 5 (Table 3).

Filter No. 1 is most suitable for sterilization. In addition to those listed, they also produce a so-called pre-filter, designed to free the filtered liquid from large particles contained in it.

Chamberlant and Berkefeld filters (candles) are hollow cylinders, closed at one end. Chamberlant candles are made from kaolin mixed with sand and quartz. They are standardized by pore size and designated L 1, L 2, L 3 ... L 13. Berkefeld filters (candles) are prepared from infusor soil; according to the size of their pores they are designated V, N, W, which corresponds to a pore diameter of 3-4, 4-7, 8-12 microns.

Work with bacterial filters is carried out as follows. The filter must be secured in a special holder, which is inserted into the filter receiver. The receiver is usually a Bunsen flask. The holders, in most cases made of stainless steel, consist of two parts: the upper, shaped like a cylinder without a bottom, and the lower, a supporting part ending in a tube. Seitz filters with the rough surface facing up are placed on metal mesh and securely clamped with screws between the top and bottom of the holder. The mounted filter is secured in a rubber stopper inserted into the neck of a Bunsen flask. A cotton swab is inserted into the outlet tube of the flask, which is connected to the vacuum pump. The prepared installation is wrapped in paper and sterilized in an autoclave under a pressure of 1 atm for 20-30 minutes. The entire assembled device is also called a Seitz filter (Fig. 13).

Immediately before filtration, the outlet end of the Bunsen flask is connected by a rubber tube to an oil or water jet pump. The junctions of the various parts are filled with paraffin to create a tight seal. The filtered liquid is poured into the cylinder of the apparatus and the pump is turned on, creating a vacuum in the receiver. As a result of the resulting pressure difference, the filtered liquid passes through the pores of the filter into the receiver, and the microbes remain on the surface of the filter.

Before use, membrane filters are sterilized by boiling in distilled water. To prevent filters from curling, they are first placed in distilled water, heated to a temperature of 50-60 ° C, and boiled over low heat for 30 minutes, changing the water 2-3 times. The filter holder and receiver are sterilized in advance, and the device is mounted under aseptic conditions. To avoid tearing the membrane filter on the metal mesh, place mugs of sterile filter paper under it. Then, using sterile tweezers with smooth tips, take the membrane filter from the sterilizer and place it on the support grid with the shiny surface down.

Candles (Chamberlant) sterilized in an autoclave are connected through a rubber tube to a receiver and lowered into a vessel (usually a cylinder) with a filtered liquid. Filtration occurs using a vacuum pump. A sterile filtrate enters the receiver, and bacteria are retained by the pores of the candle.

Membrane and asbestos filters are designed for single use. After use, candles are boiled in tap water, then calcined in a muffle furnace.

Before subsequent use, candles are checked for integrity. The candle is lowered into a vessel with water and air is passed through. If air bubbles appear on the surface of the candle, it means that cracks have formed in the candle and it is unusable.

Control questions

1. What is the filter sterilization method? What is sterilized using this method?

2. What bacterial filters do you know? How is the filtering device installed, what conditions must be observed?

Chemical methods

This type of sterilization is used to a limited extent, and it serves mainly to prevent bacterial contamination of culture media and immunobiological preparations (vaccines and serums).

To preserve vaccines and serums, merthiolate, boric acid, formaldehyde, etc. are used.

Biological sterilization

Biological sterilization is based on the use of antibiotics. This method is used for cultivating viruses.

Control questions

1. What is chemical sterilization and when is it used?

2. What is biological sterilization?

The main methods of sterilization are presented in table. 4.

1 (Sterilization is incomplete: spores remain in the sterilized material.)

2 (Sterilization is incomplete: viruses remain in the sterilized material.)

Disinfection

In microbiological practice, various disinfectants are used: 3-5% phenol solutions, 5-10% Lysol solutions, 1-5% chloramine solutions, 3-6% hydrogen peroxide solutions, 1-5% formaldehyde solutions, mercuric chloride solutions in dilution 1: 1000 (0.1%), 70° alcohol, etc.

Spent pathological material (pus, feces, urine, sputum, blood, cerebrospinal fluid) is disinfected before draining it into the sewer. Disinfection is carried out with dry bleach or 3-5% chloramine solution.

Pipettes (graduated and Pasteur), glass spatulas, slides and coverslips contaminated with pathological material or cultures of microorganisms are immersed in glass jars with a 3% solution of phenol or hydrogen peroxide for a day.

After finishing work with infectious material, the laboratory technician must treat it with a disinfectant solution. workplace and hands. The surface of the work table is wiped with a piece of cotton wool moistened with a 3% phenol solution. Hands are disinfected with a 1% chloramine solution. To do this, moisten a cotton ball or gauze with a disinfectant solution and wipe the left hand, then the right, and then wash your hands with warm water and soap.

The choice of a disinfectant, its concentration and duration of exposure (exposure) depend on the biological properties of the microbe and on the environment in which the disinfectant will come into contact with pathogenic microorganisms. For example, mercuric chloride, phenol, and alcohols are unsuitable for disinfecting protein substrates (pus, blood, sputum), since under their influence protein coagulation occurs, and the coagulated protein protects microorganisms from the effects of disinfectants.

When disinfecting material infected with spore forms of microorganisms, a 5% solution of chloramine, 1-2.5% solutions of activated chloramine, 5-10% solutions of formalin and other substances are used.

Disinfection, which is carried out throughout the day during work, is called current, and at the end of work - final.

Disinfectants and instructions for preparing working solutions from them. Chloride of lime is a white, lumpy powder with a pungent odor of chlorine; it does not completely dissolve in water. The bactericidal effect depends on the content of active chlorine, the amount of which ranges from 28 to 36%. Chlorine containing less than 25% active chlorine is unsuitable for disinfection.

If stored improperly, bleach decomposes and loses some of its active chlorine. Decomposition is promoted by heat, moisture, sunlight, so store bleach should be kept in a dry, dark place, in a tightly closed container.

Dry bleach is used to disinfect human and animal secretions (at the rate of 200 g per 1 liter of feces and 10 g per 1 liter of urine).

Preparation of the original 10% clarified bleach solution. Take 1 kg of dry bleach, place it in an enamel bucket and grind it. Then pour cold water to a volume of 10 liters, mix well, cover with a lid and leave for a day in a cool place. After this, the resulting 10% clarified solution is carefully drained and filtered through several layers of gauze or filtered through a thick cloth. Store in dark glass bottles, closed with a wooden stopper, in a cool place for no more than 10 days. Working solutions of the required concentration are prepared from the stock solution immediately before use. The amount of basic solution required for preparing 0.2-10% clarified bleach solutions is given in table. 5.

The concentration of clarified bleach solutions from 0.2 to 10% is selected depending on the nature of the object being disinfected and the resistance of the pathogen.

Chloramine is a crystalline substance of white or yellowish color, containing 24-28% active chlorine. It dissolves well in water at room temperature, so solutions are prepared immediately before disinfection. Use 0.2-10% chloramine solutions. The relationship between the percentage concentration of the solution and the amount of chloramine in grams per 1 and 10 liters is given in table. 6.

Dissolve chloramine in glass or enamel dishes. When storing chloramine solutions in dark glass containers with a ground-in stopper, their activity persists for up to 15 days.

Activated chloramine. The disinfecting properties of chloramine are enhanced by adding an activator to it in a ratio of 1:1 or 1:2. Ammonium compounds are used as an activator - ammonium chloride, sulfate, ammonium nitrate. Activated chloramine is used in concentrations of 0.5, 1 and 2.5%. They are prepared immediately before use. Chloramine and ammonium salt are weighed separately. First, chloramine is dissolved in water, and then an activator is added.

The advantage of activated chloramine solutions over conventional ones is that the addition of an activator accelerates the release of active chlorine. Therefore, the drug has a detrimental effect not only on vegetative forms of microorganisms, but also on their spores. Activated chloramine is used in lower concentrations and with less exposure.

Phenol (carbolic acid) is a colorless, needle-shaped crystal with a pungent, characteristic odor. When exposed to light, air and moisture, the crystals acquire a crimson-red color. Store in closed banks made of dark glass and in a place protected from light.

Phenol is soluble in water, alcohol, ether, and fatty oils. Possessing great hygroscopicity, it absorbs moisture from the environment and becomes liquid. Liquid carbolic acid contains 90% crystalline phenol and 10% water.

Use 3-5% aqueous solutions of carbolic acid prepared from crystalline phenol and liquid carbolic acid according to the scheme given in table. 7. The activity of phenol increases when it is dissolved in hot water (40-50° C).

Attention! Crystalline phenol or liquid carbolic acid, if it gets on the skin, can cause irritation, and in high concentrations - severe burns. Therefore, carbolic acid must be handled with great care. When preparing solutions, you should wear rubber gloves or as a last resort Lubricate your hands with Vaseline.

If carbolic acid gets on your skin, wash it off immediately with warm water and soap or 40° ethyl alcohol.

Note. To prepare disinfectant solutions of phenol, it is more convenient and safer to use liquid carbolic acid.

Control questions

1. What disinfectants are used in microbiological practice?

2. Describe appearance and the basic properties of bleach, chloramine, phenol.

3. What solutions of disinfectants are used to disinfect material infected with spore forms of microorganisms?

Exercise

Prepare 2 liters of 5% working solution of clarified bleach; 500 ml of 3% chloramine solution, 300 ml of 1% activated chloramine solution.

Attention! Before you start preparing solutions, make calculations.

Sterilization- infertility; destruction of pathogenic and non-pathogenic microorganisms in vegetative and spore forms in any material.

Preparing dishes for sterilization. Laboratory glassware must be cleanly washed and sterilized. For washing, use soap or chemical solutions detergents. New dishes are pre-boiled in a 1-2% solution of hydrochloric acid to avoid subsequent leaching of the glass. Dishes washed in running water are rinsed with distilled water and dried.

Bacteriological tubes. Conical, matte flasks are closed with cotton-gauze stoppers, consisting of tightly twisted rolls of cotton wool, covered with a layer of gauze. Metal stoppers in the form of outer caps have also been developed for bacteriological test tubes. It should be taken into account that sterilizing cotton plugs at high temperatures leads to the release of substances from the cotton wool that inhibit the growth of some sensitive bacteria, such as Brucella.

When installing pipettes, insert a cotton swab into the upper end. Pasteur pipettes must have a sealed capillary. Each measuring pipette is wrapped in a long strip of paper 4-5 cm wide, starting from the spout, in a helical manner along its entire length. Pasteur pipettes are wrapped in paper, 10-20 pieces each, test tubes - 15-20 pieces each. It is better to store all types of pipettes before and after sterilization in special metal cases. The stoppers on the flasks are additionally covered with paper caps.

Before sterilization, clean, assembled Petri dishes are wrapped in paper, 3 to 4 pieces each. After sterilization, the paper protects sterile glassware from contamination by microflora.

Before sterilization, the dishes are placed in the drying cabinet not too tightly to ensure air circulation, and care is taken that the temperature does not exceed 180? C, since at a higher temperature the paper and cotton wool will char. After sterilization is completed, the drying cabinet is not opened until then. Until the temperature in it drops to 70-80? C, because sharp drop temperatures may cause glass to break.

If the dishes are intended for sterilization of nutrient media in them by autoclaving under a pressure of at least 1 atm, then they are not pre-sterilized. When sterilizing media with flowing steam or in an autoclave under a pressure of no more than 0.5 atm. Sterile containers must be used.

Sterilization with dry heated air. The method is used to sterilize clean glassware. For this purpose, a Pasteur oven is used - a special drying cabinet with double walls. The outside is lined with heat-proof material. At the top there is a thermometer. An automatic electric heating element is placed at the bottom between the heat-proof casing and the inner metal casing. When the drying cabinet is turned on, the air inside it heats up. Once the set temperature is reached, the start time of sterilization is noted. Sterilization mode: at a temperature of 155-160? C - exposure for 2 hours, at 165-170? C - 1-1.5 hours, at 180? C - 1 hour. After the sterilization time, heating is stopped.

Autoclaving. This is steam sterilization under pressure combined with high temperature special apparatus- autoclave. When saturated steam encounters a cooler object, the steam condenses into water, releasing a large amount of heat. In addition, the volume of steam is reduced, which facilitates its penetration into the internal parts of the material being sterilized. A prerequisite is the supply of truly saturated steam, so that its contact with a cold object leads to immediate condensation and heating. The industry produces vertical and horizontal autoclaves.

A vertical autoclave is a double-walled cylindrical metal cauldron, sealed with a lid. Water is poured between the walls through a special tap with a funnel to a certain level. The inner wall of the boiler is equipped with holes in the upper part and a tap in the lower part, through which, when the water is heated, steam displaces air from the boiler. A metal protective frame is placed on top of the autoclave, and there must be free space between it and the autoclave itself. The autoclave is heated by connecting to the electrical network.

The autoclave is loaded with the material to be sterilized, the lid and the tap through which water was poured are closed, and the bottom tap is temporarily left open. The heated water between the walls of the autoclave boils, the resulting steam rises up and passes through the upper holes of the inner wall into the boiler, pushing out the air through the lower open tap. When all the air is displaced and the steam begins to come out in an even stream, the lower valve is closed. As a result, the steam pressure inside the autoclave increases. The beginning of sterilization is considered the moment when the pressure reaches a given value (according to the pressure gauge). Heat is adjusted throughout sterilization, maintaining steam pressure at the same level. If the pressure inside the autoclave increases excessively, there is a safety valve through which the excess steam automatically escapes.

As the steam pressure increases, the temperature in the autoclave increases accordingly.

The pressure gauge shows the steam pressure without taking into account the ambient atmospheric pressure (760 mm Hg). After the sterilization time has expired, the autoclave is turned off. After cooling, when the pressure gauge reading is zero, open the valve to release steam.

A horizontal autoclave differs from a vertical autoclave in design, but its operating principle is the same.

virological sterilization pathological animal

Samples of forms to be filled out when sending pathological material to the laboratory

Dry heat is used to sterilize laboratory glassware. The dishes prepared for sterilization are loosely loaded into the oven to ensure uniform and reliable heating of the material being sterilized. Close the cabinet door tightly, turn on the heating device, bring the temperature to 160-165 ° C and sterilize at this temperature for 1 hour. At the end of sterilization, turn off the heating, but do not open the cabinet door until the oven has cooled down; Otherwise, the cold air entering the cabinet may cause cracks in the hot cookware.

Sterilization in a Pasteur oven can be carried out at different temperatures and exposures (sterilization time) (Table 1).

Table 1. Sterilization mode

To determine the temperature inside the Pasteur oven, you can use sucrose or granulated sugar, which caramelizes at a temperature of 165-170 ° C.

Petri dishes are wrapped in paper one or more pieces at a time or placed in special metal cases.

Note. At high temperatures, the paper in which cups and pipettes are wrapped, and cotton wool turn yellow and can even become charred, so each new type of paper received by the laboratory should be tested at the accepted temperature conditions.

Control questions

1. What is meant by the term sterilization?

2. How is sterilization carried out?

3. What is sterilized by calcination over fire?

4. Describe the structure and operating mode of the Pasteur oven.

5. What is sterilized in a Pasteur oven?

6. How are glassware prepared for sterilization?

7. Why can’t nutrient media and rubber objects be sterilized in a Pasteur oven?

Exercise

Prepare Petri dishes, graduated pipettes, Pasteur pipettes, test tubes, flasks and vials for sterilization.